Anthrax toxin consists of three proteins secreted by Bacillus anthracis - protective antigen (PA), edema factor (EF) and lethal factor (LF). LF is a Zn2+dependent metalloprotease that cleaves members of the mitogen-activated protein kinase (MAPKK) family, blocking the MARK signaling pathway in professional phagocytes. The binary complex of PA and LF alone, termed lethal toxin or LeTx, is sufficient to induce death of target cells and kill experimental animals, manifesting the symptoms associated with anthrax. As one of the primary virulence factors in the pathogenesis of anthrax, LF has become an important therapeutic target for drug development. A number of small-molecule-based inhibitors of LF have been shown to be able to neutralize cytotoxicity of LeTx in cell cultures and lethality of LeTx in animal trials, promising potential therapeutic value in the treatment of anthrax infection. Human neutrophil a-defensins (HNPs) are members of a growing family of small cationic antimicrobial peptides that kill a broad range of microbes such as bacteria, fungi and viruses, playing an important role in the innate immune defense against infectious pathogens. Kaufmann and colleagues recently discovered that HNP1 non-competitively inhibits LF, preventing cleavage of a MAPKK and restoring impaired MAPK signaling in LeTx-treated macrophages (Kim et al., 2005). Further, HNP1 rescued murine macrophages from B. anthracis-induced cytotoxicity, and in vivo treatment with HNP1-3 protected mice against the fatal consequences of LeTx (Kim et al 2005). In a subsequent report, Mayer-Scholl et al. (2005) demonstrated that B. anthracis spores engulfed by human neutrophils and germinated intracellularly can be effectively killed by HNPs. These findings raise the possibility that a-defensins may be developed as a novel class of antitoxins to combat anthrax infection. Despite the therapeutic potential of a-defensin-based antitoxins for anthrax treatment, the molecular basis for the inhibition of LF by a-defensins remains poorly understood. In this proposal, we seek to decipher the sequence rules and structural determinants that dictate the inhibition of LF by a-defensins, and to design second-generation defensin molecules with significantly improved inhibitory activity. [unreadable] [unreadable] [unreadable]